PROJECT SUMMARY Inflammatory bowel disease (IBD), including ulcerative colitis and Crohn?s disease, affects 1.5 million people in the United States. The incidence of colitis-associated cancer (CAC), or colorectal cancer in patients with IBD, is nearly six-fold higher than that of the general population. Therefore, the morbidity and mortality of IBD is quite high. Understanding the molecular mechanisms behind IBD and CAC pathogenesis may lead to improved screening and therapeutics. Like other cancers driven by chronic inflammation, CAC is characterized by genetic mutations and aberrant gene expression programs in intestinal cells. Leucine-rich repeat-containing G protein- coupled receptor 5 (LGR5) is a membrane receptor that increases WNT signaling in intestinal stem cells, conferring self-renewal and proliferation. Furthermore, LGR5+ stem cells are thought to be the cells-of-origin of intestinal malignancy; thus, transcription of Lgr5 is tightly regulated. However, mechanisms of transcriptional regulation of Lgr5 are largely unknown. Myeloid translocation gene on chromosome 16 (MTG16) is a transcriptional co-repressor originally discovered as a translocation fusion partner in therapy-related acute myeloid leukemia. Mtg16-/- mice exhibit aberrancies in baseline intestinal phenotypes and increased susceptibility to chemical models of colitis and CAC. Preliminary data using a luciferase reporter assay and chromatin immunoprecipitation (ChIP) indicate that MTG16 represses Lgr5 expression through occupancy of intron 1 of Lgr5. Using a yeast two-hybrid screen and protein complex immunoprecipitation (Co-IP), we identified the elongation factors MLL1, AFF4, and DOT1L as potential MTG16 interacting partners. Mutation or deletion of certain Nervy homology regions (NHRs) in MTG16 abolished interaction in yeast two-hybrid growth assays and increased the severity of dextran sodium sulfate-induced colitis in vivo. Taken together, we hypothesize that specific NHRs in MTG16 bind to transcription and elongation factors to regulate Lgr5 expression, impacting homeostasis, injury, and tumorigenesis in the intestinal epithelium. This hypothesis will be tested via two focused, mechanistic, and hypothesis-driven Aims. In Specific Aim 1, the components of the endogenous MTG16 repression complex of Lgr5 in the intestine will be elucidated using Co- IP and sequential ChIP in in young adult mouse colon (YAMC) cells. Lentiviral overexpression of mutant MTG16 in a previously CRISPR-generated MTG16-deficient YAMC line will be used to determine NHR dependency of complex formation and Lgr5 repression in intestinal cells. In Specific Aim 2, novel MTG16 NHR mutant mice will be used to test for NHR dependency on intestinal epithelial homeostasis, injury, and tumorigenesis. Importantly, clarifying the role of MTG16 in intestinal epithelial biology will further our understanding of stem cell biology and may elucidate novel mechanisms for targeting pro-proliferative and tumorigenic pathways.